Tyr1 phosphorylation promotes phosphorylation of Ser2 on the C-terminal domain of eukaryotic RNA polymerase II by P-TEFb

Abstract
Data availability
Article and author information
Metrics

Abstract

The Positive Transcription Elongation Factor b (P-TEFb) phosphorylates Ser2 residues of C-terminal domain (CTD) of the largest subunit (RPB1) of RNA polymerase II and is essential for the transition from transcription initiation to elongation in vivo. Surprisingly, P-TEFb exhibits Ser5 phosphorylation activity in vitro. The mechanism garnering Ser2 specificity to P-TEFb remains elusive and hinders understanding of the transition from transcription initiation to elongation. Through in vitro reconstruction of CTD phosphorylation, mass spectrometry analysis, and chromatin immunoprecipitation sequencing (ChIP-seq) analysis, we uncover a mechanism by which Tyr1 phosphorylation directs the kinase activity of P-TEFb and alters its specificity from Ser5 to Ser2. The loss of Tyr1 phosphorylation causes an accumulation of RNA polymerase II in the promoter region as detected by ChIP-seq. We demonstrate the ability of Tyr1 phosphorylation to generate a heterogeneous CTD modification landscape that expands the CTD’s coding potential. These findings provide direct experimental evidence for a combinatorial CTD phosphorylation code wherein previously installed modifications direct the identity and abundance of subsequent coding events by influencing the behavior of downstream enzymes.

Data availability

All mass spec data generated or analyzed during this study are included in the manuscript and supporting files. Source data files have been provided in Figure 3f, Figure 1-figure supplement 2 and Figure 3-figure supplement 2 and 4.

The following data sets were generated

1. Irani S
2. Zhang Z
3. Liu Z
4. Zhang Y
(2019) RNA polymerase II ChIP with dasatinib inhibition
NCBI Gene Expression Omnibus. GSE131838.

https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE131838

Article and author information

Author details

Joshua E Mayfield

Department of Molecular Biosciences, University of Texas at Austin, Austin, United States

Competing interests
The authors declare that no competing interests exist.
Seema Irani

Department of Chemical Engineering, University of Texas at Austin, Austin, United States

Competing interests
The authors declare that no competing interests exist.
Edwin E Escobar

Department of Chemistry, University of Texas at Austin, Austin, United States

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-0086-6264
Zhao Zhang

Department of Molecular Medicine, University of Texas Health Science Center at San Antonio, San Antonio, United States

Competing interests
The authors declare that no competing interests exist.
Nathaniel T Burkholder

Department of Molecular Biosciences, University of Texas at Austin, Austin, United States

Competing interests
The authors declare that no competing interests exist.
Michelle R Robinson

Department of Chemistry, University of Texas at Austin, Austin, United States

Competing interests
The authors declare that no competing interests exist.
M Rachel Mehaffey

Department of Chemistry, University of Texas at Austin, Austin, United States

Competing interests
The authors declare that no competing interests exist.
Sarah N Sipe

Department of Chemistry, University of Texas at Austin, Austin, United States

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0003-4554-7571
Wanjie Yang

Department of Molecular Biosciences, University of Texas at Austin, Austin, United States

Competing interests
The authors declare that no competing interests exist.
Nicholas A Prescott

Department of Molecular Biosciences, University of Texas at Austin, Austin, United States

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-0635-8906
Karan Kathuria

Department of Molecular Biosciences, University of Texas at Austin, Austin, United States

Competing interests
The authors declare that no competing interests exist.
Zhijie Liu

Department of Molecular Medicine, University of Texas Health Science Center at San Antonio, San Diego, United States

Competing interests
The authors declare that no competing interests exist.
Jennifer Brodbelt

Department of Chemistry, University of Texas at Austin, Austin, United States

Competing interests
The authors declare that no competing interests exist.
Yan Zhang

Department of Molecular Biosciences, University of Texas at Austin, Austin, United States

For correspondence
jzhang@cm.utexas.edu

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-9360-5388

Funding

National Institute of General Medical Sciences (R01 GM104896)

Yan Zhang

National Institute of General Medical Sciences (R01 RM125882)

Jennifer Brodbelt

National Institute of Biomedical Imaging and Bioengineering (R21EB018391)

Jennifer Brodbelt

Welch Foundation (F-1778)

Yan Zhang

Welch Foundation (F-1155)

Jennifer Brodbelt

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.